Development of a 5´trans-splicing Gene Therapy

Dystrophic epidermolysis bullosa (DEB) is an inherited mechano-bullous skin disease caused by mutations in the COL7A1gene coding for type VII collagen. Type VII collagen is an important structural protein of the basal membrane zone (BMZ), connecting dermis and epidermis. Loss of function or lack of type VII collagen results in a separation below the lamina densa as a consequence of mechanical loads. A bullous phenotype characterised by blistering and erosions of skin and mucosa is the consequence. Because of its transcript length of 9,2kb a cDNA gene therapy for type VII collagen is difficult to establish, because this size and the repetitive nature of the COL7A1 hinders stable integration into vectors.. This project focuses on correcting mutations 5 to exon 64 of COL7A1 by SMaRT (Spliceosome mediated mRNA trans- splicing). SMaRT is a gene repair approach on RNA level and uses the naturally occurring trans-splicing process to exchange the disease causing part of a gene. The advantages in comparison to a standard gene therapy are maintenance of endogenous transcription control, the ability to target dominant negative mutations and the size reduction of the delivery vector. A PTM (pre trans-splicing molecule) which encodes the wildtype copy of the disease causing part of the target gene is introduced into the cell. The PTM is colocalized to its target by a specific binding domain. After colocalisation the trans-splicing prozess is carried out by the spliceosome and results in a wildtype mRNA. One can exchange different parts of your target gene by SMaRT. The most critical part of a PTM is its binding domain. To select the best binding domains for human COL7A1 we have developed a FACS based screen with fluorescent proteins. The screen and the constructs shall be adapted to the murine system for animal experiments. The best human and murine PTMs will be tested on their endogenous activity in a human DEB patient cell line and murine keratinocytes of a type VII collagen hypomorphe mouse. The functionality of the constructs, i.e. the recovery of wildtype type VII collagen, in DEB keratinocytes shall be tested by western blotting, RT-PCR and fluorescence staining. Corrected keratinocytes will be tested on their ability to form functional skin sheets with a stable BMZ by skin sections and flurescence staining. This should be the last step previous to animal experiments.

Dystrophic epidermolysis bullosa (DEB) is an inherited mechano-bullous skin disease caused by mutations in the COL7A1gene coding for type VII collagen. Type VII collagen is an important structural protein of the basal membrane zone (BMZ), connecting dermis and epidermis. Loss of function or lack of type VII collagen results in a separation below the lamina densa as a consequence of mechanical loads. A bullous phenotype characterized by blistering and erosions of skin and mucosa is the result. Because of its transcript length of 9,2kb a cDNA gene therapy for type VII collagen is difficult to establish, because this size and the repetitive nature of the COL7A1 hinder stable integration into vectors. This project focuses on correcting mutations 5 to exon 64 of COL7A1 by SMaRT (Spliceosome mediated mRNA trans-splicing). SMaRT is a gene repair approach on RNA level and uses the naturally occurring trans-splicing process to exchange the disease causing part of a gene. The advantages in comparison to a standard gene therapy are maintenance of endogenous transcription control, the ability to target dominant negative mutations and the size reduction of the delivery vector. A PTM (pre trans-splicing molecule) which encodes the wildtype copy of the disease causing part of the target gene is introduced into the cell. The PTM is colocalized to its target by a specific binding domain. After colocalisation the trans-splicing prozess is carried out by the spliceosome and results in a wildtype mRNA. One can exchange different parts of your target gene by SMaRT. The most critical part of a PTM is its binding domain. To select the best binding domains for human COL7A1 we have developed a FACS based screen with fluorescent proteins. The screen and the constructs were then adapted to the murine system for animal experiments. The best human and murine PTMs were tested on their endogenous activity in a human DEB patient cell line and murine keratinocytes of a collagen 7 hypomorphic mouse. The functionality of the constructs, i.e. the recovery of wildtype type VII collagen, in DEB keratinocytes was tested by western blotting, Real-Time-PCR and immunofluorescence staining. In RealTime-PCR an increase of COL7A1 mRNA from 25% of wildtype levels in patient cells to 75% of wildtype levels in treated patient was shown. In the murine experiment the increase of mRNA levels was significant as well. The result was verified on protein level by immunofluorescent staining of type VII collagen in cell culture. For two human constructs which can correct mutations within the first 64 exons of COL7A1 protein secretion was shown in Western Blot and correct localization of the recovered protein within the basal membrane zone was shown in constructed skin sheets.